Little is known about the molecular mechanism of estrogen action in normal mammary gland or about the molecular basis for the loss of estrogenic regulation which occurs in mammary cancer. We have recently observed that normal mouse mammary gland, not unlike certain human and rodent mammary neoplasias can also lose its sensitivity to estrogenic regulation. Importantly in normal tissue this phenomenon is reversible and estrogenic regulation can be fully restored. The specific aim of the proposed research is to determine if a modification the estrogen receptor (ER) system mediates the reversible loss of responsiveness to estrogenic regulation in normal mouse mammary gland. To accomplish this I intend to examine and compare estrogen receptor-hormone interactions in estrogen-sensitive and estrogen insensitive states of normal mammary glands as follows: (1) Characterize cytoplasmic estrogen receptors with respect to concentration of binding sites, binding kinetics, affinity, and steroid specificity. (2) Examine the process of receptor activation by kinetic parameters, changes in molecular size, and differences in binding affinity to DNA. (3) Examine estrogen receptor interactions with the nucleus with respect to the time course and amount of ER nuclear uptake, retention, and cytoplasmic ER replenishment. Elucidation of the role of ER in the mechanism by which normal mammary tissue can reversibly alter its responsiveness to estrogen may provide a model for delineating the molecular basis of hormonal insensitivity of human mammary tumors and may ultimately provide a basis for therapeutic reversal of hormone independence in these women.